Gram-positive bacteria, including Streptococci or Staphylococci are important pathogens and the etiological agent of a number of serious diseases including pneumonia, sepsis, meningitis, wound infections, endocarditis, acute rheumatic fever, neonatal sepsis or toxic shock syndrome. Therefore there is a need to develop vaccines against these pathogens. Vaccines are already available for some S. pneumoniae serotypes; these have shortcomings such as a highly complex manufacturing process.
S. pneumoniae is a highly diverse polysaccharide encapsulated alpha-hemolytic Streptococcus that frequently colonizes the human nasopharynx and can cause non-invasive pneumococcal diseases such as otitis media, sinusitis and non-bacteraemic pneumonia, and more severe invasive diseases such as bacteraemia/sepsis, meningitis and bacteraemic pneumonia, primarily among young children and the elderly.
The polysaccharide capsule is a major determinant of virulence during invasion and prevents C3b opsonisation and non-opsonic killing by neutrophils. Currently licensed vaccines contain capsular polysaccharide antigens formulated either alone in pneumococcal polysaccharide vaccines (PPSV) or conjugated to a carrier protein such as modified diphtheria toxin CRM 197 in pneumococcal conjugate vaccines (PCV). There are more than 90 different capsular serotypes in 40 serogroups.
Polysaccharide pneumococcal vaccines can provide serotype-specific protection but cross-protection is low even within the same serogroup. Serotype replacement has been observed after introduction of the conjugate vaccine Prevnar® 7 in the U.S. in 2000. Among the emerging serotypes are also multi-drug resistant capsule-switch variants. Therefore there is a need for next generation pneumococcal vaccines that target other antigens than the capsule.
One potential antigen for inclusion into a next generation pneumococcal vaccine is Pneumococcal Surface Protein A (PspA). PspA is a monomeric polymorphic cholin-binding protein and contains an N-terminal alpha-helical part, which forms an antiparallel coiled-coil with itself, a proline-rich region, which is sometimes interspersed by a relatively conserved non-proline block, and a C-terminal region containing multiple repeats of a choline binding domain.
The N-terminal region of PspA contains immunodominant epitopes. Recombinant proteins comprising this region and bacterial vectors expressing this region have shown protective potential in various models. For example, Langermann et al. have prepared recombinant Bacille Calmette-Guérin (rBCG) vectors expressing PspA. In order to be able to anchor the PspA in the bacterial membrane, a PspA-derived gene segment was fused to Mtb19 lipoprotein (see Langermann S. et al., J. Exp. Med. 1994, 180, 2277-2286). There is a safety concern associated with the use of PspA as a vaccine antigen because the N-terminal region may resemble human myosin and thus immunization with an immunogen encompassing this region may lead to tissue cross-reactive antibodies. Therefore recent efforts have been made to use other regions of PspA as antigen. Another PspA region that may be suitable for inclusion into a next generation pneumococcal vaccine is the proline-rich region. Although the proline-rich region (PRR) of PspA is polymorphic, it contains several conserved motifs, including short amino acid motifs like PKP, PAPAP, PEKP, and a highly conserved non-proline block (NPB) that is present in some PspA molecules (Brooks-Walter, A. et al., Infect Immun 1999, 67, 6533-6542; Hollingshead, S. K. et al., Infect Immun, 2000, 68, 5889-5900; Daniels, C. C. et al., Infect Immun, 2010, 78, 2163-2172.). Although the PRR does not contain immunodominant epitopes, antibodies against this part of PspA have been detected in children using an enzyme immunoassay (EIA) with a thioredoxin (Trx) fusion protein as antigen (Melin, M. et al., Vaccine 2012, 30, 7157-7160). Because the NPB is highly conserved the authors hypothesize that antibodies to the PRR may cross-react with a majority of strains through their recognition of NPB epitopes.
The PRR of PspA has a small size (up to around 100 amino acids) and therefore may not be sufficiently immunogenic when used as an antigen alone. Escherichia coli Trx fusion proteins have been produced and their potential for protection has been demonstrated in a mouse model of intravenous infection (WO 2007/089866 and Daniels, C. C. et al., Infect Immun, 2010, 78, 2163-2172). However, Trx fusion proteins may not be suitable for human use as a vaccine because of a potential for the induction of immune responses to non-protective Trx epitopes and poor structural representation of native PR epitopes.
Moreover NPB or proline-rich (PR) sequences may also contain non-protective epitopes, and hence it may be critical to concentrate immune responses to protective epitopes for efficacy.
Similar PR sequences can be found in other pneumococcal proteins, including the surface proteins PspC (also known as CbpA or Hic), and the PhtX proteins PhtA, PhtB, PhtD and PhtE, and proline-rich regions derived from such other pneumococcal proteins may be suitable for inclusion into a next generation pneumococcal vaccine, like proline-rich sequences from PspA.
Several immunogenic bacterial surface proteins from other Gram-positive bacteria contain proline-rich sequences that can likewise be targeted by vaccines against these pathogens. These proteins include surface proteins from other Streptococci such as the M6, SclA and SclB proteins of S. pyogenes, CBeta (bac) and BibA of S. agalactiae or the P1 adhesin of S. mutans, or proteins from S. aureus. 
Synthetic bacterial lipopeptide analogs have received wide attention in vaccine research, both for their adjuvant effects and as carriers for peptide antigens (Ghielmetti M. et al., Immunobiology 2005, 210, 211-215). Many lipopeptide constructs have been reported, in which a lipid with known adjuvant effects has been coupled to a peptide to generate self-adjuvanting vaccine candidates. Particularly well studied are tripalmitoyl-S-glyceryl cysteine (N-palmitoyl-S-(2,3-bis-(O-palmitoyloxy)-propyl)-cysteinyl- or Pam3Cys) and dipalmitoyl-S-glyceryl cysteine (2,3-bis-(O-palmitoyloxy)-propyl)-cysteinyl- or Pam2Cys). These lipid moieties are found in lipoprotein components of the inner and outer membranes of gram-negative bacteria. Patent application WO 98/07752 describes the use of lipopeptides for drug targeting purposes, wherein the peptide portion may be a collagen-like sequence capable of inducing triple helical structures. Patent application WO 2008/068017 describes synthetic virus-like particles comprising helical lipopeptide bundles and having a spherical or spheroidal structure with a lipid core and a peptidic outer surface. The peptide chain of the lipopeptides comprises a coiled-coil domain. The properties of the coiled-coil domain in the peptide chain of the lipopeptide building blocks determine the number of building blocks combining to form the synthetic virus-like particle.